Recently, the modification of nuclear, mitochondrial, and cytoplasmic proteins by O-linked 2-N- acetylglucosamine (O-GlcNAc) has emerged as a novel regulator of the stress response and cell survival. Numerous forms of cellular injury, including cardiac ischemic preconditioning (acute and prolonged), lead to elevated levels of O-GlcNAc in both in vivo and in vitro models. Elevating O-GlcNAcylation before, or immediately after, the induction of cellular injury is protective in models of ischemia reperfusion injury, as well as heat stress, oxidative stress, endoplasmic reticulum stress, hypoxia, and trauma hemorrhage. Together, these data suggest that O-GlcNAc is a novel endogenous cardioprotective agent. However, the molecular mechanisms by which O-GlcNAc regulates protein function leading to enhanced cell survival and cardioprotection have not been identified. The long-term goal of this investigator, is to identify at a molecular level the mechanisms by which O-GlcNAc promotes cell survival. The objective of this application is to: Specific Aim 1: Develop and characterize organelle specific O-GlcNAc sensors to assess the role of O-GlcNAc in ischemia reperfusion injury. We propose to develop a suite of FRET-based O-GlcNAc sensors that can map spatial and temporal changes in O-GlcNAcylation in specific subcellular compartments, and apply these sensors to determining how O-GlcNAcylation is regulated in response to ischemia reperfusion injury in isolated neonatal cardiomyocytes. Specific Aim 2: Define the dynamic site-specific O-GlcNAcome of hearts subjected to Ischemia reperfusion injury. O-GlcNAc levels are elevated in response to ischemic preconditioning, a process known to enhance cardioprotection. To identify these proteins, and the sites at which they are dynamically O-GlcNAcylated, we will develop a novel tool to enrich O-GlcNAc-modified peptides. Together, these studies will characterize a novel endogenous defense mechanism of the heart, highlighting new targets for the development of alternative strategies that enhance the hearts tolerance to ischemia reperfusion injury.